The goal of this research is to develop a small animal model for hepatitis C virus (HCV) replication by transplantation of human fetal hepatocytes to immunodeficient mice. A number of different immunodeficient mouse strains will initially be compared for susceptibility to engraftment by hepatocytes directed to the recipient liver by intrasplenic injection. Engraftment will be assessed by serological assays for human alfa-fetoprotein in mouse plasma, by immunohistochemical staining for human proteins, and by PCR for human Alu Sb2 repeats from DNA recovered from engrafted tissues. In an attempt to overcome innate "natural immunity" in immunodeficient mice, which may provide a barrier to long-term engraftment with xenogeneic tissue, the effect of recipient gamma irradiation, natural killer (NK) cell depletion and macrophage depletion on transplant survival will be evaluated. The efficacy of endogenously generated hepatocyte growth hormone (HGF) in expanding the critical mass of transplanted human tissue, and in improving HCV viremia will also be assessed. Isolated hepatocytes will be infected in vitro with virus inocula containing known numbers of HCV RNA molecules. Following transplantation of cells to appropriately conditioned immunodeficient mice, HCV replication will be assessed by reverse transcription and PCR (RT-PCR) for both positive and negative strand HCV RNA in blood and transplanted tissues, and by quasispecies analysis of the viral variants which establish infection in these animals. Finally, serial passage of virus from infected to non-infected transplanted animals will be performed to provide definitive proof of HCV replication. The availability of a reliable small animal model for HCV replication will assist in the development of anti-viral drugs and the design of a vaccine for this leading cause of chronic liver disease and hepatocellular carcinoma.